Plants show susceptibility to various stresses under both wild and cultivated conditions. In many areas of the world, high concentrations of salt in the soil are commonly encountered phenomenon. Salinity is a complex stress that involves both ionic and osmotic components. Salt stress adversely affects plant development and productivity by generating ion toxicity, inducing nutritional deficiencies, creating osmotic stress and water deficits, and decreasing photosynthesis (Shabala and Munns, 2012).

Reactive oxygen species are partly reduced forms of oxygen and by-products of aerobic metabolism and stronger oxidants than molecular oxygen. ROS are overproduced under stress condition and can potentially damage the intracellular machinery. ROS may include free radicals such as superoxide anion (O2•-) and hydroxyl radical (^∙OH), as well as non-radical molecules like hydrogen peroxide (H₂O₂) and singlet oxygen (¹O₂). Plants can scavenge a certain amount of ROS generated as by-products of aerobic metabolism, but under stress conditions, including salt stress, the resulting photoinhibition often creates an imbalance between ROS generation and scavenging due to generation of excessive amount of ROS, thereby resulting in oxidative stress, causing peroxidation of lipids, oxidation of proteins, inhibition of enzyme activity, injury to nucleic acids, activation of programmed cell death (PCD) pathways, and eventually leading to death of the cells (Gill and Tuteja, 2010; Hasanuzzaman et al., 2013). The antioxidant defense machinery is a ROS scavenging system, which protects plants from oxidative damage. Efficient enzymatic (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; glutathione reductase, GR; monodehydroascorbate reductase, MDHAR; dehydroascorbate reductase, DHAR; glutathione peroxidase, GPX; guaicol peroxidase, GOPX; and glutathione S-transferase, GST) and non-enzymatic (ascorbate, AsA; glutathione, GSH; phenolic compounds, alkaloids, non-protein amino acids, and α-tocopherols) components form an antioxidant defense system to protect overproduction of ROS and to prevent plant cells from experiencing oxidative damage (Gill and Tuteja, 2010).

Methylglyoxal (MG) is a transition-state intermediate product of the triose-phosphates of the glycolysis pathway in eukaryotic cells. High accumulation of MG is toxic and inhibits cell proliferation, causes degradation of proteins by modifying arginine, lycine, and cysteine residues, forms adducts with guanyl nucleotide in DNA, and inactivates antioxidant defense system, and causes oxidative stress (Yadav et al., 2005). The glyoxalase system comprises of two enzymes, gly I (glyoxalase I) and gly II (glyoxalase II), which catalyze the detoxification reaction of MG to D-lactate (Yadav et al., 2005). Although the glyoxalase enzymes have been extensively studied in microbial and animal systems (Thornalley, 1990), the biological significance of this pathway in plants and under stress condition is just beginning to be explored (Yadav et al., 2005).

The polyamines (PAs) are low-molecular-weight organic cations found in a wide range of organisms, where they perform diverse biological functions (Kusano et al., 2008). Putrescine (Put), spermidine (Spd), and spermine (Spm) are the most common PAs. The levels of PAs frequently increase during stress and they play roles in enhancing plant stress tolerance. Acting as molecular chaperones, PAs bind to negatively charged surfaces and protect membranes and biomolecules (Kusano et al., 2008). PAs may also act as ROS and free-radical scavengers and activate the antioxidant enzyme machinery which help to reduce oxidative stress and subsequent membrane injury and electrolyte leakage (Gill and Tuteja, 2010; Kubiś et al., 2014). PAs interact with some signaling molecules (Kusano et al., 2008). PAs have been confirmed to be unique polycationic metabolites involved in the direct blockage of a variety of cation and K⁺-selective channels, vacuolar-type channels, and ammonium channels (Dobrovinskaya et al., 1999). PAs restrict plasma membrane Na⁺ influx and NaCl-induced K⁺ efflux and shoot-to-root K⁺ recirculation (Shabala et al., 2007; Zhao et al., 2007). Despite their range of protective effects under stress condition, acute application of PAs can cause endogenous PAs catabolism in the apoplast, which is responsible for ROS-induced oxidative damage (Di Tomaso et al., 1989). The diversity of the physiological functions of PAs has made understanding the detailed mechanisms of the protective function of PAs in response to stress particularly challenging.

Salt stress tolerance is a polygenic trait. Some of the major traits affected by salinity include the maintenance of membrane transport activity and ionic homeostasis, compartmentalization of ions at the cellular and whole-plant level (which may include Na⁺ exclusion from root uptake, intra-cellular Na⁺ sequestration, cytosolic K⁺ retention), osmotic adjustment through synthesis of compatible solutes, oxidative stress tolerance, biomembrane protection, and MG toxicity tolerance (Yadav et al., 2005; Shabala and Munns, 2012; Rahman et al., 2016). Changes in ranges of biochemical pathways and the induction of hormones and signaling molecules activate stress responsive genes that impart salt stress tolerance (Adem et al., 2014). Polyamines have diverse mechanism to enhance salt tolerance. Under salt stress PAs proved to be efficient in modulating membrane integrity and ion transport processes, vacuolar ion channels, shoot, and root growth (Di Tomaso et al., 1989; Dobrovinskaya et al., 1999), photosynthesis (Kotzabasis et al., 1993; Duan et al., 2008; Shu et al., 2012), antioxidative responses (Sheokand et al., 2008; Ikbal et al., 2014); Na⁺ and nutrient influx/efflux (Shabala et al., 2007; Pottosin et al., 2012). However, the actual mechanism by which PAs modulate these physiological processes is not yet known. A few components of antioxidant system were studied in response to PAs modulation. Polyamine-induced MG detoxification process is unknown in plants. In the present study, we have investigated the roles of PAs in maintaining ionic and nutrient homeostasis, inducing osmotic adjustment, and protecting mung bean plant cells from oxidative damage and MG toxicity under salt stress. The results presented here provide new insight into the role of PAs in enhancing salt tolerance and mitigating salt stress-induced damage in mung bean seedlings by the coordinated action of PAs on ion homeostasis, antioxidant defense, and the glyoxalase system.

Polyamines play multifunctional roles to improve plant physiology both under normal growth condition and under stress condition. Reduction of Na influx, modulation of ion transport processes (Di Tomaso et al., 1989; Dobrovinskaya et al., 1999), upregulation of antioxidant system and alleviation of oxidative stress, osmotic adjustment (Sheokand et al., 2008; Ikbal et al., 2014) and improvement of photosynthesis (Kotzabasis et al., 1993; Duan et al., 2008; Shu et al., 2012) are some vital functions of PAs improving salt tolerance in plants. However, in previously conducted experiment some physiological parameters were studied but the actual mechanism by which PAs modulates these physiological processes were not executed. A few components of antioxidant system were studied under salt stress in response to PAs. Polyamine-induced MG detoxification process is unexplained in plants. Present study mainly focuses on the antioxidant system and oxidative stress in detail. We execute the MG toxicity under salt stress and find out the roles of PAs to alleviate MG toxicity. We also investigate roles of PAs in reducing Na uptake, maintaining nutrient homeostasis, osmotic adjustment, plant water status, protecting photosynthetic pigments from salt damage and overall growth performance of mung bean seedlings.

An influx of Na⁺ into root epidermal cells through plasma membrane non-selective channels, followed by a depolarization and activation of the K⁺ efflux, are the immediate and primary responses of plants exposed to salt stress (Shabala et al., 2007; Zhao et al., 2007). Salt-affected mung bean seedlings showed a higher Na accumulation and reduced K level, (Figures 1A–D). Replacement of Na⁺ by K⁺, Na⁺ exclusion, and retention of intracellular K⁺ are all important salt tolerance mechanisms (Shabala et al., 2007). An increase in K and decrease in Na level in PAs-supplemented salt-affected seedlings (Figures 1A–D) indicated the influential roles of PAs in reducing Na toxicity. Zhao et al. (2007) revealed that PAs reduced Na uptake, increased K uptake and improved K⁺/Na⁺ homeostasis by regulating root ion channel activities, thereby enhancing salt tolerance in barley seedlings. Exogenously applied PAs inhibited non-selective cation channels and decreased NaCl-induced membrane depolarization and K⁺ efflux in pea mesophyll cells (Shabala et al., 2007).

Imbalances in nutrient homeostasis were another component of the salt-induced damage in mung bean seedlings. Depolarization of root plasma membrane, disruption of plasma membrane ion channels, replacement of nutrient by excess Na influx are reasons for reduction of nutrient uptake and imbalances in nutrient homeostasis under salt stress (Shabala et al., 2007; Zhao et al., 2007). Reductions in Ca, Mg, and Zn content in roots and shoots were clearly evident in the salt-stressed seedlings (Figures 1E–J). However, exogenous Put, Spd, or Spm alleviated the negative effects of salt stress by increasing the mineral nutrient levels (except for Zn content), when compared to salt stress only (Figures 1E–J). Pottosin et al. (2012) reported that PAs improve membrane transportation, regulate movement of cation/anion through membrane and maintained nutrient homeostasis. Further clarification of PAs-induced mineral homeostasis will add value to this research topic.

Under salt stress, imposition of ionic and osmotic stress interrupts the soil-plant water relationship and reduces plant water uptake, thereby causing physiological drought (Shabala and Munns, 2012). Salt stress-induced osmotic stress is one of the primary detrimental effects that induce expression of compatible solute associated genes, de novo synthesis of compatible solutes (Shabala and Munns, 2012; Adem et al., 2014). Similar physiological drought and osmotic stress was distinguished in salt affected mung bean seedlings as indicated by reduced leaf RWC and accumulation of Pro. Exogenous PAs application to salt-stressed plants maintained the Pro level at the same level as seen in the salt only treatment, but increased the leaf RWC (Table 2). Here, together with Pro, PAs were supposed to be involved in the regulation of other osmoprotectant molecules that maintained the tissue water content/leaf RWC at a high level (Duan et al., 2008; Adem et al., 2014; Kubiś et al., 2014).

Salt increases the activity of the chlorophyllase enzyme, making the pigment–protein complex unstable, while salt-induced ROS degrade photosynthetic pigments (Shu et al., 2012). Salt stress decreased leaf photosynthetic pigments, including chl a and total chl (a+b) (compared to control) and exogenous PAs supplementation to salt-stressed plants improved chl (a+b) content (Table 2). PAs rapidly enter the chloroplast, where they protect the photosynthetic apparatus from the adverse effects of stresses (He et al., 2002). The roles of PAs might therefore be the reduction of oxidative stress, maintenance of cellular pH, protection of pigment–protein complexes, protection of thylakoid membranes, regulation of chl biosynthesis and degradation pathways, and regulation of the enzymes related to those pathways. Supplementation of PAs relaxed salt stress effects and prevented chl degradation which restored and increased the contents of leaf chl (Li et al., 2014).

Inhibition of photosynthesis results in an over-reduction of the photosynthetic electron transport chain reactions, and it redirects photon energy into processes that promote the production of ROS and oxidative damage under salt stress (Sheokand et al., 2008; El-Shabrawi et al., 2010; Gill and Tuteja, 2010; Shabala and Munns, 2012). Salt stress creates ionic toxicity which inactivates the vital enzymes involved in major physiological processes, disrupted photosystem activity and mitochondrial electron transport chain causing imbalance between production and scavenging ROS, thereby amplify oxidative stress. Salt induced physiological drought, osmotic stress and stomatal closure are also responsible for ROS overproduction (Shabala and Munns, 2012). Mung bean plants exposed to salt stress showed higher accumulations of ROS, including H₂O₂ and O2•-, and showed higher LOX activity, which resulted in cellular oxidative damage, as reflected in increases in membrane lipid peroxidation or MDA levels (Figures 2C–F). Visual identification of H₂O₂ and O2•-, detected by histochemical staining (with DAB and NBT, respectively), also reflected a similar pattern of ROS generation under salt stress (Figures 2A,B). Exogenous application of PAs reduced LOX activity, contents and spots and of H₂O₂ and O2•-, and lipid peroxidation in mung bean seedlings (Figures 2A–F). In their study, Ikbal et al. (2014) reported that PAs biosynthesis was correlated with reduction spots of H₂O₂ and O2•- in grapevine leaves. Reductions in LOX activity and reduced MDA contents under salt stress were also observed in previous studies (Nahar et al., 2015). PAs act as ROS and free-radical scavengers (Gill and Tuteja, 2010), enhance antioxidant machinery (Gill and Tuteja, 2010; Kubiś et al., 2014), PAs act as molecular chaperones and PAs bind to negatively charged surfaces and protect membranes and biomolecules (Kusano et al., 2008). Exogenous Put had a protective effect on salt-induced membrane damage in chick pea, where increased activities of antioxidant enzymes decreased ROS generation (Sheokand et al., 2008). Similar results were reported in grapevine plants where PAs biosynthesis increased significantly (Ikbal et al., 2014).

A balanced state of cellular ROS equilibrium is maintained by the interaction between enzymatic and non-enzymatic antioxidants which combat oxidative damage. Constitutes the first line enzymatic defense against ROS, SOD converts O2•- to H₂O₂, and H₂O₂ is readily detoxified by CAT by through conversion of H₂O₂ to H₂O and O₂ (Gill and Tuteja, 2010). Mung bean seedlings showed increased SOD and decreased CAT activity, acutely increased O2•- and H₂O₂ contents in salt-stressed seedlings, supported by previous studies (Nahar et al., 2016). PAs maintained same SOD and increased CAT activities (Table 1) in salt-stressed plants which overwhelmed overproduced O2•- and H₂O₂ levels and improved leaf appearance by reducing damage spots (of O2•- and H₂O₂) (Figures 2A,B; Table 1). APX, MDHAR, DHAR, and GR are the enzymes of the ascorbate-glutathione cycle which has major roles in ROS scavenging process and regenerating AsA and GSH. APX decomposes H₂O₂ via oxidation of AsA to DHA. Ascorbate reacts with O2•-, H₂O₂ to form MDHA or DHA. This reaction leads to DHA accumulation, which is harmful for plant cells. AsA is regenerated from DHA by MDHAR and DHAR, where NADPH and GSH are used as electron donors, respectively (Gill and Tuteja, 2010). Increased APX activity (Table 1) and DHA content (Figure 3B) and decreased AsA content (Figure 3A) decreased the AsA/DHA ratio (Figure 3C) of mung bean seedlings corroborating higher content of ROS. The increase in GSH level was not significant in salt-affected mung bean seedlings in the present study (compared to control), which showed increased ROS content and lipid peroxidation (Figure 2). Application of PAs increased activity of DHAR (Table 1), contents of AsA and AsA/DHA ratio, decreased DHA content (Figures 3A–C), increased the GR activity (enzyme involved in recycling of GSH) (Table 1), contents of GSH and GSH/GSSG ratio, decreased GSSG content (Figures 3D–F) which played major roles in the ROS (Figures 2D–F) detoxification process (Gill and Tuteja, 2010). Previous studies revealed the regulatory function of PAs in enhancing AsA-GSH cycle components and other antioxidant components that contributed to stress tolerance. Exogenous Spd application to salinized nutrient solution increased the antioxidant enzyme activities, including SOD, peroxidase (POD), and CAT, which alleviated salt-induced membrane damage and photosynthesis inhibition and promoted an increase in PAs content (Duan et al., 2008). Polyamine biosynthesis was correlated with enhanced APX, POD, SOD, and MDHAR activities, increased AsA content, decreased DHA levels, increased AsA/DHA ratio, increased GSH levels, decreased GSSG levels, and increased GSH/GSSG ratios in grapevine leaf tissues. Therefore, enhancement of PAs biosynthesis contributed to salt stress tolerance by upregulating ROS scavenging action and protecting the photosynthetic apparatus from oxidative damage (Ikbal et al., 2014). Using GSH as a substrate, GPX catalyzes the reduction of H₂O₂ and organic lipid hydroperoxides (Gill and Tuteja, 2010). GST conjugates GSH to a range of reactive aldehydes and xenobiotics to convert water-soluble and less toxic products. GSTs bear peroxidase activity, which reduces oxidative stress (Gill and Tuteja, 2010). The mung bean seedlings in this experiment showed a higher GPX and GST activity under salt stress, when compared to unstressed controls. GPX activity increased but GST activity did not increase further after PAs addition to salt-stressed plants, when compared to salt stress alone (Table 1). PAs have protective effects on proteins/antioxidant enzymes; PAs form complexes with SOD, GPX, and CAT for which these enzymes function more efficiently, compared with isolated enzymes (Li et al., 2014).

Methylglyoxal is a highly cytotoxic compound that causes protein and DNA degradation. MG disrupts the antioxidant machinery and act as mediator for O2•- generation, causing an oxidative burst (Yadav et al., 2005). The mung bean seedlings had high MG content under salt stress. The glyoxalase system comprises of two enzymes, Gly I, and Gly II, which catalyze the detoxification reaction of MG to D-lactate. This detoxification process occurs mainly via two steps: Gly I converts MG to SLG utilizing GSH, while Gly II converts SLG to D-lactic acid, and regenerates GSH (Yadav et al., 2005). Exogenous Put, Spd, and Spm application enhanced Gly II activity and increased the contents of GSH, which reduced MG toxicity by reducing its content (Figures 4A–C and 5D). Glutathione homeostasis and enhanced glyoxalase system activity were the biomarkers for salt tolerance in a Pokkali cultivar of rice (El-Shabrawi et al., 2010). Hasanuzzaman et al. (2011) reported that enhanced salt tolerance in wheat seedlings was partly contributed by increased activities of Gly I and Gly II. Modulation of PAs and MG in polyethylene glycol-affected white spruce was reported by Kong et al. (1998). The glyoxalase system was studied mostly in animals and microbes but not extensively in plants. It can be considered in stress specific studies.

The ratio of free [(Spd + Spm)/Put] is considered more important than the individual contents of Put, Spd, and Spm. A high [(Spd + Spm)/Put] ratio is crucial for imparting plant stress tolerance (Duan et al., 2008; Yang et al., 2010). Decreases in free Spd and Spm and increases in free Put decreased the [(Spd + Spm)/Put] ratio (Figures 5A–D), which indicates increased susceptibility of mung bean plants to NaCl toxicity (Yang et al., 2010). Therefore, the increase in Spd and Spm contents and elevation of the [(Spd + Spm)/Put] free ratio by exogenous application PAs with salt stress (Figure 5D) was critical in improving NaCl tolerance, in agreement with other published results showing that modulation of PAs was correlated with an altered physiology and biochemistry toward development of plant stress tolerance (Duan et al., 2008; Yang et al., 2010).

Higher NaCl concentration in the soil primarily causes ionic and osmotic stress that interrupts water transportation and reduces stomatal conductance, the efficiency of photosystems, RuBisCo activity, CO₂ assimilation, and photosynthesis, thereby reducing growth in salt-affected plants (Shu et al., 2012). In the present study, the overall tolerance was estimated by seedling growth and development. Exogenous application of PAs to salt-affected mung bean seedlings resulted in better seedling growth, as determined by their higher plant height, root length, and DW, when compared to salt-affected seedlings (Table 2) which is supported by previous findings. Exogenous Spd positively affected the photosynthetic and xanthophyll cycle in salt affected cucumber seedlings. Spermidine alleviated the salt-mediated decline in photosynthetic efficiency by enhancing the maximum quantum efficiency and actual efficiency of photosystem II (PSII), and improving the net photosynthetic rate, which improved overall growth performance of cucumber seedlings (Shu et al., 2012). Polyamine biosynthesis was correlated with oxidative stress protection of photosynthetic apparatus, enhancement of PSII quantum yield in grapevine plants that improved photosynthesis and plant growth (Ikbal et al., 2014).

In present study, salt stress created ionic and osmotic stress, disrupted biochemical processes, and altered PAs metabolism, resulted phytotoxicity in mung bean seedlings. High cellular Na content, imbalances in the mineral nutrients, oxidative damage, MG toxicity, and growth inhibition were the characteristic symptoms of salt stress affected mung bean seedlings. Polyamines played diversified roles in imparting salt stress tolerance in mung bean seedlings in the present study. The possible mechanism of PAs-induced salt stress tolerance has been presented in Figure 6. Exogenous PAs supplementation to salt-stressed mung bean plants modulated the endogenous levels of Put, Spd, and Spm and increased the (Spd+Spm)/Put ratio which might have regulatory roles in alteration of physiological features and stress defense mechanism of salt affected mung bean seedlings. Exogenous PAs application in mung bean seedlings increased content of AsA and AsA/DHA ratio, GSH content and GSH/GSSG ratio, enhanced activities of CAT, DHAR, GR, GPX which reduced ROS production, oxidative stress and subsequence membrane lipid peroxidation. PAs application in salt affected seedlings increased the content of GSH and activities of Gly I and Gly II enzymes which played vital roles in reducing MG toxicity. Reduction of ROS and MG by PAs were manifested through biomembrane and biomolecules protection and reduced oxidative damage in mung bean seedlings. Application of PAs prevented Na influx/toxicity and improved nutrient homeostasis in salt affected mung bean seedlings probably by regulating the plasma membrane ion channel. In salt affected mung bean seedlings, PAs increased Pro content which helped to increase leaf RWC, compared to salt stress alone. Osmoregulation and maintaining tissue water are vital for smooth running of physiological processes smoothly, stress recovery and tolerance which were imparted by PAs under salt stress. Preventing degradation, PAs improved potosynthetic pigment contents which might increase photosynthesis and growth of mung bean seedlings in present study.

KN, MH, and MF conceived and designed the experiments; KN, MA, AR, JM, and TS performed the experiments; MH analyzed the data; MF contributed reagents/materials/analysis tools; KN and MH wrote the manuscript. All authors read and approved the final manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.